Method of fixing and expressing physiologically active substance

ABSTRACT

The present invention provides methods for retaining and expressing physiologically active substances in a target tissue-specific-manner, by administering the physiologically active substances to target submucous tissue. Specifically, the present inventors demonstrated that, when physiologically active substances were directly administered into submucous tissues without using a carrier, the physiologically active substances were effectively and safely retained at the administration sites over long periods without loss and diffusion, and produced the effect acting in a reservoir-like fashion. The physiologically active substances administered as described above were demonstrated to produce the therapeutic effect without having an influence on organs other than the administered organ.

TECHNICAL FIELD

The present invention relates to methods for retaining and expressingphysiologically active substances in a target tissue-specific manner, inwhich the physiologically active substances are administered to thetarget submucous tissue.

BACKGROUND ART

At the nonclinical experiment level, it is now becoming possible totreat disease-model animals using techniques of introducing genes ofinterest, or conversely, suppressing the expression of genes of interestthrough RNA interference. In the case of “nucleic acid pharmaceuticalagents” using such a gene or an siRNA (generally termed “nucleic acid”),the nucleic acid administered to the living body needs to continuouslyproduce its effect and be retained over a long period. A critical factorin achieving the therapeutic effect of nucleic acid pharmaceuticalagents is how the drug delivery system (DDS) is designed.

Meanwhile, when nucleic acids are administered to the body as is, theyare rapidly degraded and thus fail to work. Accordingly, such nucleicacids are usually administered by using a carrier such as a viralvector, liposome, or atelocollagen as a DDS.

However, nucleic acid pharmaceutical agents have a serious disadvantagein that the carrier itself may induce an adverse immune response or suchin the body and thus not only the nucleic acid but also carrier must beassessed for its influence on the body. For example, according to areport, atelocollagen, when used as a carrier, induces a hypersensitiveimmune reaction to calf dermis derived collagen; the instruction manual(Non-patent Document 1) attached to the product named KokenAtelocollagen implant (syringe type) describes that adverse effects wereclinically found in 24 of a total of 1,192 patients.

Another problem is that, even when a carrier is used, normally, theintroduced nucleic acid can only be retained for about one week.

Prior art documents include, for example, Patent Documents 1 to 7 listedbelow. However, all of these inventions use carriers. Thus, theabove-described problems still remain unsolved.

[Patent Document 1] Japanese Patent Application Kohyo Publication No.(JP-A) 2003-516365 (unexamined Japanese national phase publicationcorresponding to a non-Japanese international publication)

-   [Patent Document 2] JP-A (Kohyo) 2005-538943-   [Patent Document 3] JP-A (Kohyo) H09-505575-   [Patent Document 4] Japanese Patent Application Saikohyo Publication    No. (JP-A) WO01-093856 (unexamined Japanese national phase    publication corresponding to a Japanese international publication-   [Patent Document 5] JP-A (Kohyo) 2005-503199-   [Patent Document 6] Japanese Patent Application Kokai Publication    No. (JP-A) 2007-119498 (unexamined, published Japanese patent    application)-   [Patent Document 7] (Granted/Registered) Japanese Patent No. 4054352-   [Non-patent Document 1] Instruction manual attached to the product    named Koken Atelocollagen implant (syringe type)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention provides methods for locally retaining andexpressing physiologically active substances at the administration site.Specifically, the present invention provides methods for retaining andexpressing physiologically active substances in a target tissue-specificmanner, in which the physiologically active substances are administeredto target submucous tissue.

Means for Solving the Problems

The present inventors conducted dedicated studies to solve theabove-described issues. Specifically, as a method of solving the aboveproblems, the present inventors evaluated establishment of a novelmethod that produces effects of physiologically active substanceswithout using carriers.

Specifically, the present inventors conducted an experiment of injectingnucleic acids, which are an embodiment of the physiologically activesubstances, into the submucous tissue of the large intestine inexperimental animals such as rats, and evaluated the effect. Commonknowledge envisaged that the solution would be rapidly lost due todiffusion or degradation. However, the present inventors obtained anunpredictable result. When the present inventors actually injectedsolutions containing nucleic acids into submucous tissues, the injectedsolutions were retained at the administration site without diffusing andproduced effects acting as a reservoir. This suggests that the submucousmicroenvironment is very special and the living body functions like acarrier by utilizing its own environment. Such a phenomenon has not beenobserved with previous intramuscular, intravascular, or intramucosaldelivery methods, and thus is specific to the submucousmicroenvironment. Specifically, the present invention discovered thatthe living body itself had sites that function as a DDS. The presentinventors thus succeeded in establishing the novel concept of “nature'sDDS” and a novel method based on it.

The submucous tissue of the intestinal tract has been shown to be muchmore effective than other matrices, for example, when pancreaticLangerhans islets are cultured in vitro (Tian X H et al. HepatobiliaryPancreat Dis Int. 4: 524, 2005). Even this shows that submucous tissueshave a very special microenvironment. In particular, the submucous pH,sugar chains, and cell composition are assumed to create an environmentthat is very suitable for maintaining nucleic acids.

Furthermore, the present inventors injected iopamidol, India ink, orsiRNA, each of which is an embodiment of the physiologically activesubstances, into the submucous tissue of the large intestine in rats andmice. The result showed that every substance was selectively retained atthe injection sites in the submucous tissue of the large intestine.

The present inventors discovered that, when siRNA was administered intothe submucous tissue of colitis model mice, the increase in expressionof the GalNAc4S-6ST gene in the large intestine was significantlysuppressed while it had no influence on other normal organs. Inaddition, it was also revealed that the suppression of GalNAc4S-6ST geneexpression in the large intestine resulted in suppression ofinflammatory activity and thus strong suppression of intestinal fibrousdegeneration. Furthermore, the histological therapeutic effect was alsodemonstrated based on the finding that the administration significantlysuppresses epithelial disruption, infiltration of inflammatory cellsinto lamina propria and submucosa, and thickening of muscular layer inthe large intestinal tissue.

As described above, the present invention demonstrated that, whennucleic acids are administered endoscopically into the submucous tissuesof the large intestine of subjects, the nucleic acids were specificallyretained at the administration site over a long period and couldcontinuously produce their effect. The present invention also enablesphysiologically active substances to produce their effects withoutassistance of a carrier, and thus has solved the previous problemassociated with the use of carriers.

The present invention relates to methods for retaining and expressingphysiologically active substances in a tissue-specific manner, in whichthe physiologically active substances are administered to the submucoustissue, and more specifically,

[1] a method for retaining and expressing a physiologically activesubstance in a target submucous tissue-specific manner, wherein thephysiologically active substance is administered into the submucoustissue;

[2] the method of [1], wherein the physiologically active substance isselected from nucleic acids, proteins, carbohydrates, lipids, orlow-molecular-weight compounds;

[3] the method of [2], wherein the nucleic acid is an siRNA; and

[4] the method of any one of [1] to [3], wherein the physiologicallyactive substance is administered in combination with a pharmaceuticallyacceptable carrier.

The present invention also provides:

[5] a method for treating or preventing a disease, which comprises thestep of retaining and expressing a physiologically active substance in asubmucous tissue-specific manner, by administering the physiologicallyactive substance to the diseased submucous tissue.

Effects of the Invention

The present invention provides a Drug Delivery System (DDS) formaintaining a physiologically active substance (for example, a nucleicacid, protein, carbohydrate, lipid, low-molecular-weight compound, etc.)in a target submucous tissue over a long period, for continuouslyproducing a physiologically active substance useful to the living body,or for continuously removing a physiologically active substance that isharmful to the living body. The present invention enables safe andeffective retention of physiologically active substances at theadministration sites over long periods without assistance of a carrier.Thus, physiologically active substances can be retained withoutconsidering side effects of carriers as before. This significantlyincreases the clinical feasibility of pharmaceutical agents usingphysiologically active substances (for example, nucleic acidpharmaceutical agents). In addition, the above-described pharmaceuticalagents have very little systemic side effects, because they specificallyproduce their effects at the injection sites. Thus, the pharmaceuticalagents are much safer than conventional methods.

Endoscopic examination (of the esophagus, stomach, and small and largeintestines) is routinely carried when diagnosing gastrointestinaldiseases. Thus, the present invention has the advantage in thatphysiologically active substances (for example, nucleic acids) can beinjected (for treatment) at the same time as diagnosis. The presentinvention is also expected to be applicable to the submucous tissues ofthe nose, subconjunctival tissue, or such, and thus enables actualclinical use of pharmaceuticals for cranial nerve diseases or ophthalmicdiseases, in which physiologically active substances have been difficultto deliver in the past. Thus the methods of the present invention have asuperior effect than the conventional local administration methods,specifically, enemas, nasal drips, and ocular instillations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show photographs of histological images 24 hours after injectionof FITC-labeled siRNA into the submucous tissues of rat largeintestines. The images show retention of the siRNA in the submucoustissues.

A: an image of the submucous tissue of the large intestine; B: an imageobserved under a fluorescence microscope after FITC staining; C: animage obtained by superimposing A with B.

FIG. 2: photographs showing retention of a substance administered intothe submucous tissue of the large intestine in normal rats. An X-rayfinding showed that iopamidol (white; within circle) was retained at aspecific site within the large intestine (left). In this figure, theupper panels show lateral view images, and the bottom panel shows amagnified image. A CT finding (right) showed that iopamidol was retainedat the injection site on the ventral side of large intestine (upperpanel) but it was undetectable on the craniad slice (lower panel).

FIG. 3 is a photograph showing retention of a substance administeredinto the submucous tissue of the large intestine in a normal mouse.Carbon particles (black) were retained within the submucous tissue ofthe large intestine.

FIG. 4 show photographs depicting retention of a nucleic acidadministered into the submucous tissue of the large intestine in anormal mouse. FITC-labeled siRNA (green) was retained at a specific sitein the large intestine. Images obtained with a fluorescence stereoscopicmicroscope; the magnification is 8.6-, 39-, or 102-fold from left.

FIG. 5 is a graph showing the kinetics of a nucleic acid administeredinto the submucous tissue of the large intestine in a normal mouse. Thegraph shows the concentrations of siRNA retained in the large intestine0.5, 3, 6, and 24 hours after injection into the submucous tissue of thelarge intestine. The rightmost bar (black) is a positive control,indicating the siRNA concentration before injection.

FIG. 6 shows a result of agarose gel electrophoresis after stabilitytest at 37° C. The upper panel shows the pattern of GalNAc4S-6ST siRNAalone; the middle panel shows the pattern of GalNAc4S-6ST siRNA treatedwith ribonuclease; the bottom panel shows atelocollagen-embeddedGalNAc4S-6ST siRNA treated with ribonuclease. The time shows theduration of reaction at 37° C.

FIG. 7 show graphs depicting the results on the effect of a nucleic acidadministered into the submucous tissue of the large intestine in dextransulfate sodium (DSS)-induced colitis model mice. In the DSS colitismodel, the siRNA injected into the submucous tissue of the largeintestine suppressed the enhanced expression of GalNAc4S-6ST in thelarge intestine. Meanwhile, the siRNA had no effect in the kidney.

FIG. 8 show graphs depicting an inflammatory activity-suppressing effectof a nucleic acid administered into the submucous tissue of the largeintestine in DSS colitis model mice. The left panel shows stoolconsistency, while the right panel shows fecal occult blood. Theadministration of siRNA into the submucous tissue of the large intestineresulted in suppression of disease activity.

FIG. 9 is a graph showing the colonic length-improving effect of anucleic acid administered into the submucous tissue of the largeintestine in DSS colitis model mice. The administration of siRNA intothe submucous tissue of the large intestine significantly suppressed thecontraction of large intestine.

FIG. 10 show photographs depicting the tissue-improving effect of anucleic acid administered into the submucous tissue of the largeintestine in DSS colitis model mice. The administration of siRNA intothe submucous tissue of large intestine significantly suppressed theepithelial disruption, inflammation, and fibrosis associated withcolitis. HE stain; 100-fold magnification.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors discovered that by directly administeringphysiologically active substances into submucous tissues, thesephysiologically active substances are retained at the administrationsites over a long period without loss or diffusion, and exert effectsacting as a reservoir.

The present invention provides methods for retaining and expressingphysiologically active substances in a target tissue-specific manner, inwhich the physiologically active substances are administered to thesubmucous tissue.

Herein, “mucous” (“mucosa”) more specifically refers to the macroscopicmembrane covering the surface of the lumen of hollow organs such as thegastrointestinal tract, respiratory and urogenital systems, includingmembranes covering the surface of auditory tubes, the middle ear cavityconnecting to the airway, and bulbar and palpebral conjunctivae.Normally, epithelium is the outermost layer, and the underlayer islamina propria of the mucous membrane.

As used herein, “submucous tissue (tela submucosa)” refers to the tissueunder the lamina propria that supports the epithelium. In actualclinical practice, however, a “submucous tissue” may be distinguisheddepending on a physician's “feeling”, and thus the subepithelial tissuemay be taken as “intramucosal tissue” and deeper tissue between themucosa and muscle may be identified as “submucous tissue”.

In the present invention, the “submucous tissue” is not particularlylimited, as long as its properties (pH, sugar chains, cellularcomposition, etc.) are appropriate to maintain the physiologicallyactive substances of the present invention. Such submucous tissuesinclude, for example, those of the intestinal tract, eye, ear, nose,uterus, urinary bladder, or oral cavity, subcutaneous tissues, or such,but are not limited to these examples.

In particular, the submucous tissues of the intestinal tract have beendemonstrated to be much more effective than other matrices whenpancreatic Langerhans islets are cultured in vitro (Tian X H et al.Hepatobiliary Pancreat Dis Int. 4: 524, 2005). This also suggests thatthe submucous tissues of the intestinal tract may have a very specialmicroenvironment. More specifically, in a preferred embodiment of thepresent invention, submucous tissues include those of the intestinaltract.

Such “submucous tissues of the intestinal tract” include, for example,those of intestinal tracts such as the esophagus, stomach, duodenum,small intestine, appendix, large intestine, and rectum. In a preferredembodiment of the present invention, submucous tissues of the intestinaltract include submucous tissue of the large intestine.

Herein, “physiologically active substance” is a general name forchemical substances that exert various biological effects in livingorganisms, and is not particularly limited. In the present invention, aphysiologically active substance is preferably selected, for example,from nucleic acids, proteins, carbohydrates, lipids, orlow-molecular-weight compounds.

“Nucleic acids” used in the present invention refer to both RNAs andDNAs. Chemically synthesized nucleic acid analogs, such as so-called“PNAs” (peptide nucleic acids) or Morpholino antisense oligos, are alsoincluded in the nucleic acids of the present invention. PNAs are nucleicacids in which the fundamental backbone structure of nucleic acids, thepentose-phosphate backbone, is replaced by a polyamide backbone withglycine units, and Morpholino antisense oligos are nucleic acids inwhich the pentose-phosphate backbone is replaced by a morpholinobackbone. PNAs and morpholino antisense oligos have a three-dimensionalstructure quite similar to that of nucleic acids.

For suppressing the expression of objective (target) genes, nucleicacids of the present invention include, for example, antisense nucleicacids against transcripts of target genes or portions thereof, nucleicacids with the ribozyme activity of specifically cleaving transcripts oftarget genes, and nucleic acids with the activity of using RNAi effectto inhibit the expression of target genes.

Methods for suppressing the expression of specific endogenous genesusing antisense technology are well known to those skilled in the art.There are a number of causes for the action of antisense nucleic acidsin suppressing target gene expression, including:

inhibition of transcription initiation by triplex formation;

transcription inhibition by hybrid formation at a site with a local openloop structure generated by an RNA polymerase;

transcription inhibition by hybrid formation with the RNA beingsynthesized;

splicing inhibition by hybrid formation at an intron-exon junction;

splicing inhibition by hybrid formation at the site of spliceosomeformation;

inhibition of transport from the nucleus to the cytoplasm by hybridformation with mRNA;

translation initiation inhibition by hybrid formation at the cappingsite or poly(A) addition site;

inhibition of translation initiation by hybrid formation at thetranslation initiation factor binding site;

inhibition of translation by hybrid formation at the ribosome bindingsite adjacent to the initiation codon;

inhibition of peptide chain elongation by hybrid formation in thetranslational region of mRNA or at the polysome binding site of mRNA;and

inhibition of gene expression by hybrid formation at the protein-nucleicacid interaction sites (Hirashima and Inoue, Shin Seikagaku Jikken Koza2 (New Courses in Experimental Biochemistry 2), Kakusan (Nucleic Acids)IV: “Idenshi no Fukusei to Hatsugen (Gene replication and expression)”,Ed. The Japanese Biochemical Society, Tokyo Kagakudojin, 1993, pp.319-347). There are causes for the action of antisense RNA insuppressing target gene expression includes inhibition of geneexpression by RNAi effect of double-stranded RNA formation by hybridformation with mRNA, and such. Thus, antisense nucleic acids inhibit theexpression of target genes by inhibiting various processes, such astranscription, splicing, and translation.

The antisense nucleic acids may inhibit the expression and/or functionof target genes, based on any of the actions described above. In oneembodiment, antisense sequences designed to be complementary to anuntranslated region adjacent to the 5′ end of an mRNA for a target genemay be effective for inhibiting translation of the gene. Sequencescomplementary to a coding region or 3′-untranslated region can also beused. Thus, the antisense nucleic acids to be used in the presentinvention include not only nucleic acids comprising sequences antisenseto the coding regions, but also nucleic acids comprising sequencesantisense to untranslated regions of target genes. Such antisensenucleic acids to be used are linked downstream of adequate promoters andare preferably linked with transcription termination signals on the 3′side. Nucleic acids thus prepared can be introduced into desired animals(cells) using known methods. The sequences of the antisense nucleicacids are preferably complementary to a target gene or portion thereofthat is endogenous to the animals (cells) to be transformed with them.However, the sequences need not be perfectly complementary, as long asthe antisense nucleic acids can effectively suppress expression of agene. The transcribed RNAs preferably have 90% or higher, and mostpreferably 95% or higher complementarity to target gene. To effectivelyinhibit target gene expression using antisense nucleic acids, theantisense nucleic acids are preferably at least 15 nucleotides long, andless than 25 nucleotides long. However, the lengths of the antisensenucleic acids of the present invention are not necessarily limited tothe lengths mentioned above, and they may be 100 nucleotides or more, or500 nucleotides or more.

Expression of the above-mentioned target genes can also be inhibitedusing ribozymes or ribozyme-encoding DNAs. Ribozymes refer to RNAmolecules with catalytic activity. There are various ribozymes withdifferent activities. Among others, studies that focused on ribozymesfunctioning as RNA-cleaving enzymes have enabled the design of ribozymesthat cleave RNAs in a site-specific manner Some ribozymes have 400 ormore nucleotides, such as group I intron type ribozymes and M1 RNA,which is comprised by RNase P, but others, called hammerhead and hairpinribozymes, have a catalytic domain of about 40 nucleotides (Koizumi, M.and Otsuka, E., Tanpakushitsu Kakusan Koso (Protein, Nucleic Acid, andEnzyme) 1990, 35, 2191).

For example, the autocatalytic domain of a hammerhead ribozyme cleavesthe sequence G13U14C15 at the 3′ side of C15. Base pairing between U14and A9 has been shown to be essential for this activity, and thesequence can be cleaved when C15 is substituted with A15 or U15(Koizumi, M. et al., FEBS Lett. 1988, 239, 285; Koizumi, M. and Otsuka,E., Tanpakushitsu Kakusan Koso (Protein, Nucleic Acid, and Enzyme) 1990,35, 2191; and Koizumi, M. et al., Nucl. Acids Res. 1989, 17, 7059).

In addition, hairpin ribozymes are also useful. Such ribozymes are foundin, for example, the minus strand of satellite RNAs of tobacco ringspotviruses (Buzayan, J. M., Nature 1986, 323, 349). It has been shown thattarget-specific RNA-cleaving ribozymes can also be created from hairpinribozymes (Kikuchi, Y. and Sasaki, N., Nucl Acids Res. 1991, 19, 6751;and Kikuchi, Y. Kagaku to Seibutsu (Chemistry and Biology) 1992, 30,112). Thus, the expression of the above-described target genes can beinhibited by using ribozymes to specifically cleave the genetranscripts.

Furthermore, in a preferred embodiment, nucleic acids of the presentinvention include nucleic acids with the inhibition activity of usingRNAi effect (siRNA).

The expression of target genes can be suppressed by RNA interference(hereinafter abbreviated as “RNAi”), using double-stranded RNAscomprising a sequence the same as or similar to a target gene sequence.

RNAi is a phenomenon where an mRNA comprising a base sequencecomplementary to a double-stranded RNA is degraded. RNAi is a methodbased on this phenomenon, in which the expression of an arbitrary geneis suppressed by artificially introducing a 21- to 23-merdouble-stranded RNA (small interfering RNA; siRNA). In 1998, Fire et al.discovered using C. elegans that double-stranded RNA silences genes in asequence-specific manner (Fire, A. et al., Nature 1998, 391, 806-811).After elucidating the underlying mechanism of mRNA cleavage by 21- to23-mer processed double-stranded RNA (Zamore P D. et al., Cell 2000,101, 25-33), identifying RNA-induced silencing complex (RISC) (Hammond,S. M. et al., Science 2001, 293, 1146-1150), and cloning Dicer(Bernstein, E. et al., Nature, 409, 363-366), Elbashir et al.demonstrated in 2001 that siRNA could also suppress expression in asequence-specific manner in mammalian cells (Elbashir S M. et al.,Nature 2001, 411, 494-498). Thus, application of RNAi to gene therapy isexpected.

Nucleic acids with inhibitory activity based on RNAi effect aregenerally referred to as siRNAs or short hairpin RNAs (shRNAs). RNAi isa phenomenon in which, when cells or such are introduced with shortdouble-stranded RNAs (hereinafter abbreviated as “dsRNAs”) comprisingsense RNAs that comprise sequences homologous to the mRNAs of a targetgene, and antisense RNAs that comprise sequences homologous a sequencecomplementary thereto, the dsRNAs bind specifically and selectively tothe target gene mRNAs, induce their disruption, and cleave the genetranscript, thereby effectively inhibiting (suppressing) target geneexpression. For example, when dsRNAs are introduced into cells, theexpression of genes with sequences homologous to the RNAs is suppressed(the genes are knocked down). As described above, RNAi can suppress theexpression of target genes, and is thus drawing attention as a methodapplicable to gene therapy, or as a simple gene knockout methodreplacing conventional methods of gene disruption, which are based oncomplicated and inefficient homologous recombination. The RNAs to beused in RNAi are not necessarily perfectly identical to the target genesor portions thereof; however, the RNAs are preferably perfectlyhomologous to the genes or portions thereof.

The targets of the siRNAs to be designed are not particularly limited,as long as they are regions of target genes. Any region of the gene canbe a candidate for a target.

The double-stranded RNA described above may also be closed at one endwith a hairpin structure (shRNAs). shRNAs are RNA molecules with astem-loop structure, since a portion of the single strand constitutes astrand complementary to another portion. Thus, molecules capable offorming an intramolecular double-stranded RNA are also included in thesiRNAs.

For example, even double-stranded RNAs with a structure having adeletion or addition of one or a small number of bases are included inthe siRNAs of the present invention, as long as they have the functionof suppressing the expression of target genes by RNAi effect.

Some details of the RNAi mechanism still remain poorly understood, butit is known that an enzyme called “DICER” (a member of the RNase IIInuclease family) binds to a double-stranded RNA and degrades it in tosmall fragments, called “siRNAs”. The double-stranded RNAs of thepresent invention that have RNAi effect include such double-strandedRNAs prior to being degraded by DICER. Specifically, since even longRNAs that have no RNAi effect when intact can be degraded into siRNAswhich have RNAi effect in cells, the length of the double-stranded RNAsof the present invention is not particularly limited.

For example, long double-stranded RNAs covering the full-length or nearfull-length mRNA of a target gene can be pre-digested, for example, byDICER, and then the degradation products can also be used. Thesedegradation products are expected to contain double-stranded RNAmolecules with RNAi effect. With this method, it is not necessary tospecifically select the regions expected to have RNAi effect. In otherwords, it is not necessary to accurately determine regions with RNAieffect in the mRNAs of the genes described above.

The siRNAs of the present invention are not necessarily single pairs ofdouble-stranded RNAs directed to target sequences, but may be mixturesof multiple double-stranded RNAs directed to regions that cover thetarget sequence. The siRNAs of the present invention include so-called“siRNA cocktails”.

All nucleotides in the siRNAs of the present invention do notnecessarily need to be ribonucleotides (RNAs). Specifically, one or moreof the ribonucleotides constituting the siRNAs of the present inventionmay be replaced with corresponding deoxyribonucleotides. The term“corresponding” means that although the sugar moieties are structurallydifferently, the nucleotide residues (adenine, thymine (uracil),guanine, or cytosine) are the same. For example, deoxyribonucleotidescorresponding to ribonucleotides with adenine refer todeoxyribonucleotides with adenine. The term “or more” described above isnot particularly limited, but preferably refers to a small number ofabout two to five ribonucleotides.

The siRNAs of the present invention include, for example, the siRNAs ofSEQ ID NOs: 1 and/or 2.

In another embodiment, nucleic acids aiming at suppressing theexpression of a target gene includes microRNAs (miRNAs), aptamers, andlocked nucleic acids (LNAs) generated by modifying oligonucleotides.

In the present invention, such nucleic acids are not limited to theexamples described above, and it is possible to use nucleic acids thatare appropriate for the purposes.

In addition, the “protein” of the present invention refers to a polymercomprising several amino acids, and includes not only polypeptides butalso oligopeptides. Herein, polypeptides include bothnaturally-occurring polypeptides without modification and modifiedpolypeptides. Such modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent bonding of flavin, covalentbonding of heme moiety/moieties, covalent bonding of nucleotides ornucleotide derivatives, covalent bonding of lipids or lipid derivatives,covalent bonding of phosphatidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cystine, formation of pyroglutamate,formylation, γ-carboxylation, glycosylation, formation of GPI anchors,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

For inhibiting (suppressing) the function of a (target) protein,proteins of the present invention include, for example, a target proteinvariants, which are dominant negative for the target protein, andantibodies that bind to the target protein.

“A target protein variant that is dominant negative for a targetprotein” refers to a protein that, when expressed a gene encoding theprotein, has the function of reducing or eliminating the activity of theendogenous wild type protein.

Moreover, antibodies that bind to the target protein can be prepared bymethods known to those skilled in the art. Polyclonal antibodies can beobtained, for example, by the following procedure: small animals such asrabbits are immunized with a natural target protein or a recombinanttarget protein expressed in microorganisms such as E. coli as a fusionprotein with GST, or a partial peptide thereof. Sera are obtained fromthese animals and purified by, for example, ammonium sulfateprecipitation, Protein A or G columns, DEAE ion exchange chromatography,affinity columns coupled with the target protein or a synthetic peptideto prepare antibodies. Monoclonal antibodies can be obtained, forexample, by the following procedure: small animals such as mice areimmunized with a target protein or a partial peptide thereof. Spleensare removed from the mice and crushed to isolate cells. The cells arefused with mouse myeloma cells using a reagent such as polyethyleneglycol. Clones producing antibodies that bind to a target protein isselected from among the resulting fused cells (hybridomas). The obtainedhybridomas are then transplanted in the peritoneal cavities of mice, andascites collected. The obtained monoclonal antibodies can be purifiedby, for example, ammonium sulfate precipitation, Protein A or G columns,DEAE ion exchange chromatography, affinity columns coupled with thetarget protein or a synthetic peptide.

The forms of above-described antibodies are not particularly limited aslong as they bind to a target protein. The antibodies of the presentinvention may include human antibodies, humanized antibodies created bygene recombination, fragments or modified products of such antibodies,in addition to the polyclonal and monoclonal antibodies described above.

The target proteins used as sensitizing antigens to prepare antibodiesare not limited in terms of the animal species from which the proteinsare derived. However, the proteins are preferably derived from mammals,for example, mice and humans Human-derived proteins are particularlypreferred. The proteins to be used as sensitizing antigens may be wholeproteins or partial peptides thereof. Such partial peptides of theproteins include, for example, amino (N)-terminal fragments and carboxyl(C)-terminal fragments of the proteins. Herein, “antibodies” refer toantibodies that react with a full-length protein or fragment thereof.

In addition to immunizing nonhuman animals with antigens to obtain theabove hybridomas, human lymphocytes, for example, EB virus-infectedhuman lymphocytes, can be sensitized in vitro with the proteins or withcells expressing the proteins, or with lysates thereof, and thesensitized lymphocytes can be fused with human-derived myeloma cellswith the ability to divide permanently, for example, U266, to obtainhybridomas that produce desired human antibodies with binding activityto the proteins.

When using the prepared antibodies for human administration (antibodytherapy), the antibodies are preferably human or humanized antibodies inorder to reduce immunogenicity.

In another embodiment of the present invention, proteins include, forexample, enzymes; and their forms are not particularly limited.

Herein, “carbohydrates” include all of monosaccharides,oligosaccharides, and polysaccharides. The carbohydrates of the presentinvention also include complex carbohydrates in which the abovesaccharides are covalently linked to proteins or lipids, and glycosidesin which the reducing groups of monosaccharides or oligosaccharides arelinked to an aglycon such as alcohol, phenol, saponin, or a pigment.

Herein, “lipids” include all of simple lipids, complex lipids, andderived lipids.

Herein, “low-molecular-weight compounds” include chemically synthesizedsubstances with a low molecular weight, typically ranging from aboutseveral hundreds to thousands. For inhibiting (suppressing) the functionof a target protein, it is possible to use, for example,low-molecular-weight substances that bind to the target protein. Suchlow-molecular-weight substances that bind to a target protein may benatural or artificial compounds. In general, the compounds can beobtained or produced by methods known to those skilled in the art.

In another embodiment of the present invention, the physiologicallyactive substances are not limited, and include, for example, singlecompounds such as natural compounds, organic compounds, inorganiccompounds; and compound libraries, expression products of genelibraries, cell extracts, cell culture supernatant, products offermenting microorganisms, extracts of marine organisms, and plantextracts. Each of the above-described physiologically active substancesmay be used alone or in combination with other physiologically activesubstances.

In the present invention, the physiologically active substances arepreferably used (administered) in an unmodified, non-labeled form(occasionally herein referred to as “naked form”).

The physiologically active substances of the present invention can beused after labeling, if needed. Such labels include, for example,radioisotope labels and fluorescent labels. The labels are notparticularly limited, and include alkaline phosphatase labels,peroxidase labels, biotin-labeled/streptavidin-conjugated enzymes(alkaline phosphatase, peroxidase, and such), and fluoresceinisothiocyanate (FITC).

Furthermore, the physiologically active substances of the presentinvention can be used in combination with pharmaceutically acceptablecarriers. Herein, the “pharmaceutically acceptable carriers” include,for example, vectors that are typically used as gene therapy vectors.

The above-described vectors include, for example, viral vectors such asretroviral vectors, adenoviral vectors, and adeno-associated viralvectors, and non-viral vectors such as liposomes and atelocollagen.Using the vectors, the physiologically active substances of the presentinvention can be administered by ex vivo and in vivo methods.

Other than the carriers described above, the carriers of the presentinvention include, but are not limited to, for example, water,physiological saline, phosphate buffered saline, polyvinyl alcohol,polyvinylpyrrolidone, carboxylvinyl polymer, sodium alginate,water-soluble dextran, pectin, xanthan gum, gum Arabic, gelatin, agar,glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin,stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol,and lactose. The physiologically active substances of the presentinvention may further comprise additives such as preservatives. Thephysiologically active substances of the present invention may furthercomprise other pharmacological ingredients.

The physiologically active substances of the present invention arepreferably parenteral preparations; liquid preparations such assolutions and suspensions are preferred, including, for example,injections. Other preferred dosage forms include, for example, linimentsand coating agents which are applied onto or coat the surface ofindwelling devices such as balloons and stents.

The physiologically active substances of the present invention can beadministered to subjects (patients or such) by methods known to thoseskilled in the art, such as using injection into target submucoustissues, or application or coating onto the surface of indwellingdevices such as balloons and stents. If needed, devices such asendoscopes may be used to administer the physiologically activesubstances.

The applied dose of a physiologically active substance of the presentinvention varies depending on the body weight and age of the subject(patient or such), administration method, and the like; however, theoptimum dose can be appropriately selected by those skilled in the art.

The present invention also provides methods for treating or preventingdiseases, which comprise the step of retaining and expressing thephysiologically active substances in a target tissue-specific manneraffected with the diseases, by administering the physiologically activesubstances to the submucous tissues with diseases.

The above-described “diseases” include those associated with mucosa(specifically, inflammatory bowel disease and Crohn's disease), fibroticdiseases, arthritis (osteoarthritis and rheumatoid arthritis),Alzheimer's disease, organ transplant toxicity and rejection, cachexia,allergy, cancer (for example, solid tumors/cancers including colon,breast, prostate, and brain, and malignant hematopoietic tumorsincluding leukemia and lymphoma), tissue ulceration, restenosis,periodontal diseases, bullous epidermolysis, osteoporosis, loosening ofartificial joint implants, atherosclerosis (including divulsion ofatherosclerosis leison), aortic aneurysm (including abdominal aneurysmand cerebral aneurysm), congestive heart failure, myocardial infarct,seizure, cerebral ischemia, head injury, spinal cord injury,neurodegenerative disorders (acute and chronic), autoimmune diseases,Huntington's chorea, Parkinson's disease, migraine, depression,peripheral neuropathy, pain, cerebral amyloid angiopathy, nootropic orcognitive enhancement, amyotrophic lateral sclerosis, multiplesclerosis, ophthalmovascular formation, conical injury, maculardegeneration, abnormal healing of traumatic injury, and burns; however,the diseases are not limited to the above examples, and include otherdiseases as long as the methods of the present invention are applicableto them.

Preferred subjects to be administered with the physiologically activesubstances of the present invention are mammals including humans, anddomestic animals, pets, and experimental animals. In particular, mammals(patients) with a disease described above are preferred subjects in thepresent invention.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

EXAMPLES

Hereinbelow, the present invention is specifically described usingExamples; however, it is not to be construed as being limited thereto.

Example 1

DSS enteritis was induced by allowing 12-week-old male Wistar rats tofreely drink water containing 3% dextran sulfate sodium (DSS) (molecularweight; 50,000) (Okayasu I, Hatakeyama S, Ohkusa T, Inagaki Y, Nakaya R.A novel method in the induction of reliable experimental acute andchronic ulcerative colitis in mice. Gastroenterology 1990, 98: 694-702).An ultrathin endoscope for humans was inserted into the large intestinesof rats anesthetized with Nembutal on day 0 and 3 after the start offeeding with DSS water. After observation of the mucosa, siRNA wasinjected into the submucous tissue at equally spaced four sites in theleft colon. The ultrathin endoscope used was a prototype model (outerdiameter of the scope, 5.6 mm) having a working forceps channel (channeldiameter, 2 mm) which had been developed as an upper gastrointestinalendoscope for humans by OLYMPUS. Untreated rats were used as a control.

The therapeutic effect was evaluated based on: (1) clinical diseaseactivity index (CDAI) determined in terms of the three items: weightloss, diarrhea, and melena; (2) intestinal length; and (3)pathohistological analysis of HE stained samples prepared using sectionof large intestine, in the treated and control groups.

Example 2 Retention of Physiologically Active Substances in theSubmucous Tissue of Rat Large Intestine

Next, the retention of physiologically active substances in thesubmucous tissue of normal rats was analyzed by diagnostic imaging usingX-ray and CT, which are routinely used clinically. Twelve-week-old maleWistar rats were anesthetized with Nembutal, and then an ultrathinendoscope for humans was inserted into the large intestines and 20 μl ofiopamidol, a contrast medium, was endoscopically injected alone into thesubmucous tissue of the left colon using a local injection needle.

Iopamidol refers to the compound namedN,N′-Bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2S)-2-hydroxypropanoylamino]-2,4,6-triiodoisophthalamide(C17H22I3N3O8; molecular weight, 777.09) represented by formula (I):

X-ray photography and CT scanning was carried out after one hour. Theresults are shown in FIG. 2. The X-ray images showed that the injectediopamidol was retained in the submucous tissue of the colon withoutintraperitoneal leakage and transfer to other sections. The CT imagesalso showed that the injected iopamidol was retained specifically at theinjection sites and did not spread to other slices. Thus, the clinicalevaluation method also demonstrated that the methods of the presentinvention could retain physiologically active substances veryspecifically within target submucous tissues.

Example 3 Retention of Physiologically Active Substances in theSubmucous Tissue of Mouse Large Intestine

Next, the retention of physiologically active substances was assessedusing mice, which are more common experimental animals. Eight-week-oldfemale C57BL/6J mice were anesthetized with Nembutal and laparotomizedto expose the lower part of the large intestines. 20 μl of carbonparticles (India ink), corresponding to a physiologically activesubstance of the present invention, alone was macroscopically injectedto the submucous tissues. Then, the abdomen was closed.

Five days after, the mice were sacrificed to prepare tissue sections ofthe large intestine. The sections were observed under a lightmicroscope. The result is shown in FIG. 3. The injected carbon particleswere found to be retained within the submucous tissue without physicaldiffusion and leakage to other portions.

Example 4 Local Retention of Nucleic Acid within Mouse the LargeIntestine

20 μl of FITC-labeled siRNA (20 μM) of BLOCK-iT Fluorescent Oligo(Invitrogen) was injected into the submucous tissue of mouse largeintestine by the same method as described in Examples 1 and 3. Theretention of the injected siRNA was assessed under a fluorescentstereomicroscope (Leica) 24 hours after injection.

The results are shown in FIG. 4. The FITC-labeled siRNA was confined atthe injection site in the large intestine. FIG. 1 of Example 1 is ahistological image obtained by injecting the same FITC-labeled siRNAinto the submucous tissue of rat large intestine. Based on the findingsdescribed above, the same result is predicted to be obtained when thelabeled siRNA is injected into the submucous tissue of mouse largeintestine.

From the result described above, from all different points of view usingclinical index (X-ray and CT), microscopic indicators in livingsubjects, and histological index, it was demonstrated that, wheninjected into the submucous tissue of large intestine by the method ofthe present invention, physiologically active substances, includingsiRNA, were selectively retained within the submucous tissue.

Example 5 Nucleic Acid Kinetics in the Submucous Tissue of Mouse LargeIntestine

Next, in order to also confirm the retention of injected siRNA in thesubmucous tissue of the large intestine by biochemical methods, theconcentration of siRNA in the large intestine was determined by usingthe ELISA method for direct quantitation of siRNA according to apublished report (Rosie Z et al. Analytical Biochem. 304: 19-25, 2002).The nucleotide sequences of GalNac 4S-6ST siRNA used in this Example areshown below, but the sequences are not limited to the example shownherein.

[human GalNac4-6ST siRNA] (Gene Bank accession number NM_015892)

(Hokkaido System Science Co., Ltd.)

(SEQ ID NO: 1) 5′-ggagcagagcaagaugaauacaauc-ag-3′ (SEQ ID NO: 2)3′-ua-ccucgucucguucuacuuauguuag-5′

GalNAc4S-6ST siRNA (0.3 μg/10 μl) was injected at three sites to thesubmucous tissue of large intestine in eight-week-old female C57BL/6Jmice by the same method described in Example 3. The mice were sacrificed0.5, 3, 6, or 24 hours after injection to prepare histological samplesof the large intestine. The siRNA concentrations were determined by theELISA methods.

The result is shown in FIG. 5. 1 μg/ml siRNA before injection was usedas a positive control without any additional treatment. At the timepoint after 0.5 hour, the siRNA was retained at a very highconcentration, while the concentration was decreased by half after threehours. However, the result shows that siRNA continually remainedretained 24 hours later.

Thus, not only by diagnostic imaging and histology but alsobiochemically, it was proven that the physiologically active substancesare effectively retained at the injection sites by the methods of thepresent invention.

Example 6 Stability Test for GalNAc4S-6ST siRNA

In this Example, GalNAc4S-6ST siRNA, the same substance used in Example5, was assessed for its stability. First, test solutions were preparedby adding 1 μg of GalNAc4S-6ST siRNA to 200 μl of sterile phosphatebuffer or 0.1% atelocollagen and stirring the mixtures at 4° C. for 20minutes. 0.1% atelocollagen was prepared by combining 1% atelocollagen(Koken) with ten volumes of sterile phosphate buffer and stirring themixture at 4° C. for 16 hours. Then, 40 μg of a ribonuclease (RNase A;SIGMA) was added to the prepared test solutions of GalNAc4S-6ST siRNA,and the mixtures were incubated to react at 37° C. The reaction time was5, 15, 30, 45, or 60 minutes. 500 μl of RNA iso (Takara Bio) was addedto the test solutions after reaction. The mixtures were incubated on icefor five minutes, and centrifuged at 14,000 rpm for 15 minutes. Theresulting supernatants were collected, and 500 μl of isopropanol and 1μl of glycogen (Invitrogen) were added thereto. The mixtures wereincubated for 15 minutes, and centrifuged at 14,000 rpm for 15 minutes.The supernatants were discarded, and the pellets were saved. 1 ml of 75%ethanol was added to the pellets, and the mixtures were centrifuged at14,000 rpm for 15 minutes. After this step was repeated twice, thepellets were dried and dissolved in 25 μl of injection solvent (OtsukaPharmaceutical). 10 μl each of the solutions were combined with 2 μl ofLoading Dye (Invitrogen). 3.5% agarose gel was prepared using UltraPureAgarose (Invitrogen), and the samples were electrophoresed in a Mupid-2plus (ADVANCE) at 100 V for 20 minutes. After electrophoresis, the gelwas shaken for 20 minutes in a stain solution prepared by 10,000 timesdiluting Ethidium Bromide (Invitrogen) with 1× LoTE (composition: 3 mMTris-HCl (pH 7.5) (Invitrogen), 0.2 mM EDTA (pH 7.5) (Sigma AldrichJapan)). The gel was photographed with Fluourchem (Innotech) andanalyzed.

As seen in FIG. 6, in the absence of ribonuclease, GalNAc4S-6ST siRNAwas relatively stable even after 60 minutes. However, in the presence ofribonuclease, siRNA degradation was observed at the time point after 15minutes and the band disappeared after 30 minutes. On the other hand,when GalNAc4S-6ST siRNA was embedded in atelocollagen, the band wasfaint but detectable even after 30 minutes. Short-chain RNA (siRNA)embedded in atelocollagen has been reported to be resistant toribonuclease. An equivalent result was obtained in this Example.Furthermore, the present invention also suggests that the same result isobtained not only when siRNA is embedded in atelocollagen but also inother biological substances having the same characteristics.

Example 7 Efficiency of Gene Knockdown by siRNA Administered into theSubmucous Tissue of Large Intestine in Colitis Model Mice

A colitis model mice was prepared by allowing C57BL/6J mice (female, sixweeks old; CLEA Japan Inc.) to freely drink high-concentration chlorinewater containing 3% dextran sulfate sodium (DSS; Wako Pure ChemicalIndustries Inc.) for five days. This DSS-induced colitis model hasexcellent reproducibility, and is thus used widely as a standardexperimental model for inflammatory bowel diseases such as mouseulcerative colitis and Crohn's disease (Sasaki N, J Inflamm. 2005 2: 13;as a review, Pucilowska J B et al. Am J Physiol Gastroenterol LiverPhysiol. 279: G653-G659, 2000).

The same GalNAc4S-6ST siRNA (0.3 μg/head) as used in Example 5 wasinjected to the submucous tissue of mouse large intestine, while themice were allowed to drink water containing 3% DSS. The control groupsused were: a group injected with scramble siRNA (“BLOCK-iT FluorescentOligo (Invitrogen)” in Example 4) and an untreated group in which micewere allowed to drink DSS water only. The body weight and diseaseactivity index (DAI) score (Kihara M, Gut. 2003 52: 713-9) were recordedfor five days while the mice were allowed to drink water containing 3%DDS. Then, the mice were sacrificed on the fifth day.

1 ml of RNA iso (Takara Bio) was added to 50 mg each of excised organs(large intestine and kidney). The organs were crushed using anelectrical homogenizer (DIGITAL HOMOGENIZER; AS ONE), then, 200 μl ofchloroform (Sigma-Aldrich Japan) was added to the resulting suspension.The mixture was gently mixed and then cooled on ice for about fiveminutes, and centrifuged in a centrifuge (Centrifuge 5417R; Eppendorf)at 12,000 rpm and 4° C. for 15 minutes. After centrifugation, 500 μl ofthe supernatant was transferred to a fresh eppendorf tube, and an equalvolume of isopropanol (500 μl; Sigma-Aldrich Japan) was added thereto.The solution was mixed, and then 1 μl of glycogen (Invitrogen) was addedthereto. The mixture was cooled on ice for 15 minutes, and thencentrifuged at 12,000 rpm and 4° C. for 15 minutes. Next, RNAprecipitate obtained after washing three times with 1,000 μl of 75%ethanol (Sigma-Aldrich Japan) was air-dried for 30 minutes to one hour,and then dissolved in Otsuka distilled water (Otsuka Pharmaceutical Co.,Ltd). The solution was 100 times diluted with Otsuka distilled water.The RNA concentrations of extracted samples in UV plates (CorningCostar) were determined using a plate reader (POWER Wave XS; BIO-TEK).

Next, reverse transcription reaction (cDNA synthesis) is conducted bythe following procedure. The concentrations of the obtained RNA sampleswere adjusted to 500 ng/20 μl. The samples were heated at 68° C. forthree minutes in a BLOCK INCUBATOR (ASTEC), and cooled on ice for tenminutes. After cooling on ice, 80 μl of RT PreMix solution (composition:18.64 μl of 25 mM MgCl2 (Invitrogen), 20 μl of 5× Buffer (Invitrogen),6.6 μl of 0.1 M DTT (Invitrogen), 10 μl of 10 mM dNTP mix (Invitrogen),2 μl of RNase Inhibitor (Invitrogen), 1.2 μl of MMLV ReverseTranscriptase (Invitrogen), 2 μl of Random primer (Invitrogen), and19.56 μl of sterile distilled water (Otsuka distilled water; OtsukaPharmaceutical Co., Ltd.)), which had been prepared in advance, wasadded to the samples. The mixtures were heated in a BLOCK INCUBATOR(ASTEC) at 42° C. for one hour and at 99° C. for five minutes, and thencooled on ice. 100 μl of desired cDNAs were prepared and quantitativePCR reaction was carried out using the prepared cDNAs in the followingcomposition. For quantitative PCR, SYBR Premix Kit (TAKARA) andReal-time PCR thermal cycler DICE (TAKARA) were used. Conditions of PCRreaction was: 95° C. for 10 seconds, 40 cycles of 95° C. for 5 secondsand 60° C. for 30 seconds, finally, melting curve analysis wasconducted. Nucleotide sequences of primers used in the quantitative PCRwere described below.

[Quantitative PCR Primer Sequence]

*mouse GalNac4S-6ST (TAKARA) forward: (SEQ ID NO: 3)5′-GTGAGTTCTGCTGCGGTCCA-3′ reverse: (SEQ ID NO: 4)5′-AGTCCATGCTGATGCCCAGAG-3′ *mouse GAPDH (TAKARA) Forward:(SEQ ID NO: 5) 5′-CTGCCAAGTATGACATCA-3′ Reverse: (SEQ ID NO: 6)5′-TACTCCTTGGAGGCCATGTAG-3′

The results are shown in FIG. 7. The expression of GalNAc4S-6ST wassignificantly increased in the large intestine; however, the injectedGalNAc4S-6ST siRNA significantly suppressed the increased expression.Meanwhile, in this model, the expression of GalNAc4S-6ST was notincreased in the kidney, and remained unchanged even after injection ofGalNAc4S-6ST siRNA. In addition, in the group injected with scramblesiRNA, there was no significant change in the expression ofGalNAc4S-6ST. Thus, the present invention demonstrated that GalNAc4S-6STsiRNA injected to the submucous tissue of large intestine wasselectively retained in the large intestine. Furthermore, from thefunctional viewpoint, the GalNAc4S-6ST siRNA was demonstrated tosignificantly suppress the increase in the expression of GalNAc4S-6ST inthe large intestine. The result described above suggests that thepresent invention is very effective for the retention and function ofthe injected physiologically active substances at the administrationsites, and produces no side effect on other normal organs.

Example 8 Modification of Clinical/Pathological Features by GeneKnockdown Resulting from siRNA Administered to the Submucous Tissue ofLarge Intestine in Colitis Model Mice

Since the retention and function of siRNA was demonstrated, its actualtherapeutic effect was evaluated next. The colitis activity index (DAI)was analyzed by the same method described in Example 7.

The criteria for DAI evaluation are shown in Table 1 below.

TABLE 1 WEIGHT STOOL FECAL INDEX LOSS CONSISTENCY OCCULT BLOOD 0 NONORMAL NORMAL 1  1-5% FECAL OCCULT BLOOD(+) 2 5-10% LOOSE STOOL FECALOCCULT BLOOD(++) 3 10-20%  FECAL OCCULT BLOOD(+++) 4  >20% DIARRHEASIGNIFICANT HEMORRHAGE

The first day (day 0) of feeding with DSS water is defined as 1, and thestool consistency and fecal occult blood in each mouse were recorded.The results are shown in FIG. 8. As compared to the control group, thescore was lower in the group administered with GalNAc4S-6ST siRNA. Thisresult suggests that the GalNAc4S-6ST siRNA injected into the submucoustissue of large intestine suppresses the expression of GalNAc4S-6STgenein a colon-specific manner and exerted the effect of suppressing theinflammatory activity.

Example 9 Colonic-Contraction-Improving Effect of Gene Knockdown bysiRNA Administered to the Submucous Tissue of Large Intestine in ColitisModel Mice

Next, the length of the large intestine was measured after sacrificingmice on day five. Colonic contraction was significantly suppressed inthe group administered with GalNAc4S-6ST siRNA (p<0.05; t test) (FIG.9). The length of the large intestine is a crucial indicator thatreflects the intestinal fibrosis. Thus, the GalNAc4S-6ST siRNA injectedinto the submucous tissue of large intestine was demonstrated tosuppress the expression of GalNAc4S-6ST gene in a colon-specific manner.In addition, from the clinical viewpoint, the siRNA was demonstrated tostrongly suppress fibrotic degeneration of the intestine.

Example 10 Histological Improvement Effect of Gene Knockdown by siRNAAdministered to the Submucous Tissue of Large Intestine in Colitis ModelMice

Next, tissue sections were prepared from collected large intestines andstained with hematoxylin-eosin. The resulting histological images wereanalyzed (FIG. 10). The epithelial disruption, infiltration ofinflammatory cells into lamina propria and submucosa, and thickening ofmuscular layer were markedly suppressed in the group administered withGalNAc4S-6ST siRNA. Thus, the GalNAc4S-6ST siRNA injected into thesubmucous tissue of large intestine was demonstrated to suppress theexpression of GalNAc4S-6ST gene in a colon-specific manner. In addition,the siRNA was also histologically demonstrated to produce a therapeuticeffect.

The invention claimed is:
 1. A method of treating an inflammatory bowel disease, comprising: directly injecting a formulation comprising siRNA into a target site of a submucous tissue of large intestine in a subject such that the siRNA suppresses expression of a targeted sequence, wherein the formulation comprises the siRNA in a naked form without any viral vector, and the siRNA comprises sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and suppresses expression of GalNAc4S-6ST gene.
 2. The method of claim 1, wherein the inflammatory bowel disease is ulcerative colitis.
 3. The method of claim 1, wherein the inflammatory bowel disease is Crohn's disease.
 4. The method of claim 1, wherein the directly injecting is carried out by an endoscope.
 5. The method of claim 2, wherein the directly injecting is carried out by an endoscope.
 6. The method of claim 3, wherein the directly injecting is carried out by an endoscope.
 7. The method of claim 1, wherein the inflammatory bowel disease is one of ulcerative colitis and Crohn's disease.
 8. The method of claim 7, wherein the directly injecting is carried out by an endoscope. 